This invention relates to the production of biotin using a genetically engineered organism.
Biotin (vitamin B.sub.8 or vitamin H), a coenzyme for carboxylation and decarboxylation reactions, is an essential metabolite for living cells. Exogenous biotin is required for most higher organisms; however many bacteria synthesize their own biotin.
The enzymatic steps involved in the biotin synthetic pathway from pimelyl-CoA (PmCoA) to biotin have been elucidated in Escherichia coli and Bacillus sphaericus (FIG. 1; reviewed in Perkins and Pero, Bacillus subtilis and other Gram-Positive Bacteria, ed. Sonenshein, Hoch, and Losick, Amer. Soc. of Microbiology, pp. 325-329, 1993). The steps include the conversions of 1) pimelyl-CoA to 7-keto-8-amino pelargonic acid (7-KAP or KAPA) by 7-KAP synthetase (bioF); 2) 7-KAP to 7,8-diamino-pelargonic acid (DAPA) by DAPA aminotransferase (bioA); 3) DAPA to dethiobiotin (DTB) by DTB synthetase (bioD); and 4) DTB to biotin by biotin synthetase (bioB). Synthesis of PmCoA reportedly involves different enzymatic steps in different microorganisms. The E. coli genes involved in steps preceding pimelyl-CoA synthesis include bioC (Otsuka et al., J. Biol. Chem. 263:19577-19585 (1988)) and bioH (O'Regan et al., Nucleic Acids Res. 17:8004 (1989)). In B. sphaericus, two different genes, bioX and bioW, are thought to be involved in PmCoA synthesis. BioX is thought to be involved in pimelate biosynthesis (Gloeckler et al., Gene 87:63-70, 1990), and bioW has been shown to encode pimelyl-CoA synthetase which converts pimelic acid (PmA) to PmCoA (Ploux et al., Biochem. J. 287:685-690, 1992). Neither B. sphaericus gene, bioW or bioX, has significant sequence similarity with the E. coli bioC and bioH genes either at the nucleotide or protein level (Gloeckler et al., 1990, supra).
In E. coli, the biotin biosynthetic genes are located in three or more operons in the chromosome. The bioA gene is located in one operon and the bioBFCD genes are located in a second closely linked operon. The bioH gene is unlinked to the other bio genes (FIG. 2; Eisenberg, M. A. 1987 in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1, Amer. Soc. Micro. Wash. D.C.).
In B. sphaericus, the organization of the bio genes is clearly different from that in E. coli. Gloeckler et al. (1990, supra) have isolated and characterized two unlinked DNA fragments from B. sphaericus that encode bio genes. One fragment contains an operon encoding the bioD, bioA, bioY, and bioB genes, and the other fragment contains an operon encoding the bioX, bioW and bioF genes (FIG. 2). The order and clustering of bio genes is different in E. coli and B. sphaericus (FIG. 2).
Fisher U.S. Pat. No. 5,110,731 provides a system for producing biotin wherein the genes of the biotin operon of E. coli are transformed into, and expressed in, a retention-deficient strain of E. coli.
Gloeckler et al. U.S. Pat. No. 5,096,823 describes genes involved in the biosynthesis of biotin in B. sphaericus: bioA, bioD, bioF, bioC, and bioH. B. sphaericus genes for bioA and bioD were cloned into both E. coli and B. subtilis. The bioA and bioD genes were stably integrated into B. subtilis Bio.sup.- auxotrophs, and prototrophic strains were selected.
GB 2,216,530-B2 (Jul. 8, 1992; Minister of Agr & Fisheries) provides plasmids containing gene(s) for E. coli bioA, bioB, bioC, bioD, and bioF isolated from other E. coli genetic material, e.g., control sequences. The plasmids are capable of replicating and being expressed in non-E. coli strains, preferably in yeast.
Three biotin synthesis deficient mutants of B. subtilis (bioA, bioB, and a gene termed bio112 which may be analogous to E. coli bioF) have been reported (Pai, Jour. Bact. 121:1-8, 1975; and Gloeckler et al., 1990, supra).
Nippon Zeon Co. Ltd. U.S. Pat. No. 4,563,426 discloses biotin fermentation that includes adding pimelic acid after culturing for about 24 hours. Transgene SA and Nippon Zeon Co. Ltd. E.P. 0 379 428 discloses adding pimelic acid to a biotin fermentation medium.